Normal bone development, including craniofacial development, is largely dependent on the normal differentiation of mesenchymal precursor cells into mature bone forming osteoblasts. Moreover, normal osteoblast differentiation is necessary for the production of bone matrix and the maintenance of bone integrity. Disruptions in the balance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption during bone remodeling lead to osteoporotic bone loss. This osteoporotic bone loss occurs in many areas of the body including the spine, long bones, and the craniofacial bones, resulting in debilitating bone fractures and tooth loss. Although much is known about the role of mature osteoblasts in bone formation, very little is known about the genetic and cellular events which lead to the differentiation of osteoblast precursors into mature osteoblasts. Therefore, the focus of this proposal is the isolation and characterization of genes which may be involved in the regulation of osteoblast differentiation. The proposed studies will involve a class of genes known as helix-loop-helix (HL) genes, which code for transcription factors that are known to regulate cell differentiation. Serving as a model system for the studies are the hFOB 1.19 cell line, a new human fetal osteoblastic (hFOB) cell line which is conditionally immortalized with a temperature sensitive mutant of the SV40 large T antigen. This cell line which was recently developed in our laboratory (1) is the first human fetal osteoblastic cell line of its kind. As is the case with rat fetal calvarial cultures, subconfluent, relatively undifferentiated (pre-osteoblast-like) hFOB cells differentiate upon reaching confluence in culture and mineralize the extracellular matrix. Preliminary data presented in this application show that five putative HLH cDNA fragments have been isolated and sequenced from hFOB mRNA. Further, the steady state mRNA levels of one of these genes, HLH 330, increase after hFOB cells reach confluency. The specific aims of these studies are to: (1) isolate and sequence HLH cDNA fragments from hFOB RNA, (2) characterize the pattern of expression of the HLH genes in hFOB cells, and (3) determine if the disruption of the expression of the HLH genes alters the normal differentiation pattern of hFOB cells.